Aberrant Assembly of RNA Recognition Motif 1 Links to Pathogenic Conversion of TAR DNA-binding Protein of 43 kDa (TDP-43)

Aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) is a pathological signature of amyotrophic lateral sclerosis (ALS). Although accumulating evidence suggests the involvement of RNA recognition motifs (RRMs) in TDP-43 proteinopathy, it remains unclear how native TDP-43 is converted to pathogenic forms. To elucidate the role of homeostasis of RRM1 structure in ALS pathogenesis, conformations of RRM1 under high pressure were monitored by NMR. We first found that RRM1 was prone to aggregation and had three regions showing stable chemical shifts during misfolding. Moreover, mass spectrometric analysis of aggregated RRM1 revealed that one of the regions was located on protease-resistant β-strands containing two cysteines (Cys-173 and Cys-175), indicating that this region served as a core assembly interface in RRM1 aggregation. Although a fraction of RRM1 aggregates comprised disulfide-bonded oligomers, the substitution of cysteine(s) to serine(s) (C/S) resulted in unexpected acceleration of amyloid fibrils of RRM1 and disulfide-independent aggregate formation of full-length TDP-43. Notably, TDP-43 aggregates with RRM1-C/S required the C terminus, and replicated cytopathologies of ALS, including mislocalization, impaired RNA splicing, ubiquitination, phosphorylation, and motor neuron toxicity. Furthermore, RRM1-C/S accentuated inclusions of familial ALS-linked TDP-43 mutants in the C terminus. The relevance of RRM1-C/S-induced TDP-43 aggregates in ALS pathogenesis was verified by immunolabeling of inclusions of ALS patients and cultured cells overexpressing the RRM1-C/S TDP-43 with antibody targeting misfolding-relevant regions. Our results indicate that cysteines in RRM1 crucially govern the conformation of TDP-43, and aberrant self-assembly of RRM1 at amyloidogenic regions contributes to pathogenic conversion of TDP-43 in ALS.     

Analysis of ciliary band formation in the mollusc Ilyanassa obsoleta

Two primary ciliary bands, the prototroch and metatroch, are required for locomotion and in the feeding larvae of many spiralians. The metatroch has been reported to have different cellular origins in the molluscs Crepidula fornicata and Ilyanassa obsoleta, as well as in the annelid Polygordius lacteus, consistent with multiple independent origins of the spiralian metatroch. Here, we describe in further detail the cell lineage of the ciliary bands in the gastropod mollusc I. obsoleta using intracellular lineage tracing and the expression of an acetylated tubulin antigen that serves as a marker for ciliated cells. We find that the I. obsoleta metatroch is formed primarily by third quartet derivatives as well as a small number of second quartet derivatives. These results differ from the described metatrochal lineage in the mollusc C. fornicata that derives solely from the second quartet or the metatrochal lineage in the annelid P. lacteus that derives solely from the third quartet. The present study adds to a growing body of literature concerning the evolution of the metatroch and the plasticity of cell fates in homologous micromeres in spiralian embryos.     

Cultured human corneal epithelial stem/progenitor cells derived from the corneal limbus

Stem/progenitor cells of the human corneal epithelium are present in the human corneal limbus, and several corneal epithelial stem/progenitor cell markers have been reported. Recently, the neurotrophin family receptors were reported to be useful markers of corneal epithelial stem/progenitor cells. Therefore, we examined an enzymatic separation method for obtaining corneal epithelial stem/progenitor cells and measuring the change in the expression of low-affinity neurotrophin receptor p75 (p75^NTR), a receptor belonging to the neurotrophin family. As a result, it was found that our separation method preserved cell viability. Furthermore, p75^NTR was mainly observed in epithelial basal cells as were the corneal epithelial stem/progenitor markers p63 and integrin β1. p75^NTR was also observed in the cultured cells, but its frequency decreased with passage. In conclusion, we propose that our culture method will enable the culture of corneal stem cells and that it is a useful tool for elucidating the molecular basis of the niche that is necessary for the maintenance of epithelial stem cells in the corneal limbus. Furthermore, we conclude that p75^NTR is a useful cell marker for evaluating the characteristics of stem/progenitor cells in culture.     

Secondary embryonic axis formation by transplantation of D quadrant micromeres in an oligochaete annelid

Among spiral cleaving embryos (e.g. mollusks and annelids), it has long been known that one blastomere at the four-cell stage, the D cell, and its direct descendants play an important role in axial pattern formation. Various studies have suggested that the D quadrant acts as the organizer of the embryonic axes in annelids, although this has never been demonstrated directly. Here we show that D quadrant micromeres (2d and 4d) of the oligochaete annelid Tubifex tubifex are essential for embryonic axis formation. When 2d and 4d were ablated the embryo developed into a rounded cell mass covered with an epithelial cell sheet. To examine whether 2d and 4d are sufficient for axis formation they were transplanted to an ectopic position in an otherwise intact embryo. The reconstituted embryo formed a secondary embryonic axis with a duplicated head and/or tail. Cell lineage analyses showed that neuroectoderm and mesoderm along the secondary axis were derived from the transplanted D quadrant micromeres and not from the host embryo. However, endodermal tissue along the secondary axis originated from the host embryo. Interestingly, when either 2d or 4d was transplanted separately to host embryos, the reconstituted embryos failed to form a secondary axis, suggesting that both 2d and 4d are required for secondary axis formation. Thus, the Tubifex D quadrant micromeres have the ability to organize axis formation, but they lack the ability to induce neuroectodermal tissues, a characteristic common to chordate primary embryonic organizers.     

Evaluation of mutation effects on UGT1A1 activity toward 17beta-estradiol using liquid chromatography-tandem mass spectrometry

Mutations in the gene encoding UDP-glucuronosyltransferase 1A1 (UGT1A1) may reduce the glucuronidation of estradiol, bilirubin, etc. In the present study, we used a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method to assay the activities of recombinant mutated UGT1A1 toward 17beta-estradiol (E2), by determining its glucuronide (E2G) content. Direct evidence for glucuronide formation was provided by E2G-specific ion peaks. The UGT1A1 activities of G71R (exon 1), F83L (exon 1), I322V (exon 2) and G493R (exon 5) mutants were 24, 30, 18 and 0.6% of the normal UGT1A1 activity, respectively. In conclusion, our study showed that LC/MS/MS enabled accurate evaluation of the effects of mutations on recombinant UGT1A1 activity towards E2.     

Conserved Acidic Amino Acid Residues in a Second RNA Recognition Motif Regulate Assembly and Function of TDP-43

Accumulating evidence suggests that pathogenic TAR DNA-binding protein (TDP)-43 fragments contain a partial RNA-recognition motif domain 2 (RRM2) in amyotrophic lateral sclerosis (ALS)/frontotemporal lobar degeneration. However, the molecular basis for how this domain links to the conformation and function of TDP-43 is unclear. Previous crystal analyses have documented that the RRM2-DNA complex dimerizes under acidic and high salt conditions, mediated by the intermolecular hydrogen bonds of Glu246-Ile249 and Asp247-Asp247. The aims of this study were to investigate the roles of Glu246 and Asp247 in the molecular assembly of RRM2 under physiological conditions, and to evaluate their potential use as markers for TDP-43 misfolding due to the aberrantly exposed dimer interface. Unexpectedly, gel filtration analyses showed that, regardless of DNA interaction, the RRM2 domain remained as a stable monomer in phosphate-buffered saline. Studies using substitution mutants revealed that Glu246 and, especially, Asp247 played a crucial role in preserving the functional RRM2 monomers. Substitution to glycine at Glu246 or Asp247 induced the formation of fibrillar oligomers of RRM2 accompanied by the loss of DNA-binding affinity, which also affected the conformation and the RNA splicing function of full-length TDP-43. A novel monoclonal antibody against peptides containing Asp247 was found to react with TDP-43 inclusions of ALS patients and mislocalized cytosolic TDP-43 in cultured cells, but not with nuclear wild-type TDP-43. Our findings indicate that Glu246 and Asp247 play pivotal roles in the proper conformation and function of TDP-43. In particular, Asp247 should be studied as a molecular target with an aberrant conformation related to TDP-43 proteinopathy.     

Behavior of Psychrotrophic Lactic Acid Bacteria Isolated from Spoiling Cooked Meat Products

Three kinds of lactic acid bacteria were isolated from spoiling cooked meat products stored below 10°C. They were identified as Leuconostoc mesenteroides subsp. mesenteroides, Lactococcus lactis subsp. lactis, and Leuconostoc citreum. All three strains grew well in MRS broth at 10°C. In particular, L. mesenteroides subsp. mesenteroides and L. citreum grew even at 4°C, and their doubling times were 23.6 and 51.5 h, respectively. On the other hand, although the bacteria were initially below the detection limit (<10 CFU/g) in model cooked meat products, the bacterial counts increased to 10⁸ CFU/g at 10°C after 7 to 12 days.     

Transglutaminase 2 accelerates neuroinflammation in amyotrophic lateral sclerosis through interaction with misfolded superoxide dismutase 1

Although the aberrant assembly of mutant superoxide dismutase 1 (mSOD1) is implicated in the pathogenesis of familial amyotrophic lateral sclerosis (ALS), the molecular basis of superoxide dismutase 1 (SOD1) oligomerization remains undetermined. We investigated the roles of transglutaminase 2 (TG2), an endogenous cross‐linker in mSOD1‐linked ALS. TG2 interacted preferentially with mSOD1 and promoted its oligomerization in transfected cells. Purified TG2 directly oligomerized recombinant mutant SOD1 and the apo‐form of the wild‐type SOD1 proteins in a calcium‐dependent manner, indicating that misfolded SOD1 is a substrate of TG2. Moreover, the non‐cell‐autonomous effect of extracellular TG2 on the neuroinflammation was suggested, since the TG2‐mediated soluble SOD1 oligomers induced tumor necrosis factor‐α, interleukin‐1β, and nitric oxide in microglial BV2 cells. TG2 was up‐regulated in the spinal cord of pre‐symptomatic G93A SOD1 transgenic mice and in the hypoglossal nuclei of mice suffering nerve ligation. Furthermore, inhibition of spinal TG2 by cystamine significantly delayed the progression and reduced SOD1 oligomers and microglial activation. These results indicate a novel role of TG2 in SOD1 oligomer‐mediated neuroinflammation, as well as in the involvement in the intracellular aggregation of misfolded SOD1 in ALS.